Chromatin Immunoprecipitation (ChIP) in Schizosaccharomyces pombe.
Cold Spring Harb Protoc
; 2016(11)2016 11 01.
Article
em En
| MEDLINE
| ID: mdl-27803257
ABSTRACT
Chromatin immunoprecipitation (ChIP), the cross-linking of chromatin followed by immunoprecipitation with antibodies against a chromatin target, is a key method for measuring association of proteins with a specific genomic region(s). As a negative control, a mock ChIP experiment in which no antibody is added to the immunoprecipitation reaction is included. Enriched DNA fragments from a ChIP experiment can be analyzed in a variety of ways. For semiquantitative analysis, a region of interest can be amplified using standard polymerase chain reaction (PCR) techniques. PCR products are analyzed on agarose (or polyacrylamide) gels and band intensity calculated with a standard imaging software. ChIP enrichment is usually calculated as the ratio of ChIP to input compared with a similar ratio for a reference region not expected to be enriched for that factor. For heterochromatin analysis, housekeeping genes such as act1+ are good references. Real-time quantitative PCR (qPCR) can be used for a fully quantitative approach. If a factor is to be mapped across the genome, ChIP DNA can be amplified and labeled for microarray analysis or scrutinized on a next-generation DNA sequencing platform.
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Base de dados:
MEDLINE
Assunto principal:
Schizosaccharomyces
/
DNA Fúngico
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Imunoprecipitação da Cromatina
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Proteínas de Ligação a DNA
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article